Isolated single smooth muscle cells from the fundus of a guinea-pig stomach were permeabilized by use of streptolysin-O (0.5 U/ml). Most of the permeabilized cells responded to 0.6 μM Ca 2 + , but not to 0.2 μM Ca 2 + , with a resulting maximal cell shortening to approximately 71% of the resting cell length. These cells were relaxed again by washing with the Ca 2 + -free solution (2.5 nM free Ca 2 + ) for 3-5 min. Addition of 10 μM acetylcholine (ACh) resulted in both a marked decrease in the concentration of Ca 2 + required to trigger a threshold response and an increase in the maximal cell shortening, indicating that the cells retained the muscarinic receptor function. When the cell treated with a protein kinase C (PKC) inhibitor, K-252b (1 μM), for 3 min was exposed to 10 μM ACh in the presence of K-252b, the cell shortened within 2 min with a maximal cell shortening. When the cell shortening was induced by 10 μM ACh plus 1 μM Ca 2 + in the presence of K-252b (1 μM) or more selective PKC inhibitors, such as calphostin C (1 μM) or PKC pseudosubstrate peptide (100 μM), the extension of the shortened cells, by washing with the Ca 2 + -free solution, was significantly inhibited. In contrast, K-252b (1 μM) did not inhibit the relaxation of Ca 2 + -induced shortened cells. These results suggest that the receptor-mediated activation of PKC in the process of ACh-induced cell shortening plays a role in the subsequent relaxation of the shortened cells.